Synapses between primary afferents and dorsal horn neurons are key sites for pain modulation, because they determine the intensity of nociceptive signals entering the spinal cord. NMDA receptors (NMDARs) are expressed by primary afferents and are present in their central and peripheral terminals. Although little is known about their role in pain, NMDARs in the central terminals may be involved in long-term potentiation of these synapses, thus contributing to central sensitization. This project is based on our results indicating that these NMDARs are normally in a non-functional state and become functional during the induction of chronic pain. This resolves an ongoing controversy: an initial report found that intrathecal injections of NMDA to rats induced substance P release from primary afferents, but it could not be replicated by later studies including our own. We now find that intrathecal NMDA preceded by brain-derived neurotrophic factor (BDNF) elicited a substantial amount of substance P release. Co-injection of a Src family kinase (SFK) inhibitor with BDNF suppressed the effect of NMDA, suggesting that BDNF induces the SFK phosphorylation of NMDARs. In addition to BDNF, ephrinB2 also appears to enable NMDA-induced substance P release. Importantly, we found that in a nerve injury model these NMDARs become functional for 3 days, consistent with evidence that nerve injury induces BDNF release from activated microglia and activates the ephrinB/EphB receptor system. Accordingly, this project will test the following hypotheses: 1) To be functional, NMDARs in primary afferents require Tyr-phosphorylation of their NR2B subunit, which is determined by the balance of activities of SFKs and protein tyrosine phosphatases, 2) SFK phosphorylation of NMDARs in primary afferents is initiated by BDNF and ephrins, and 3) NMDARs in primary afferent terminals are non-functional (i.e. not phosphorylated) under normal conditions and become phosphorylated in some chronic pain states. The Specific Aims are: 1) study the role of SFK phosphorylation in modulating NMDARs in primary afferents, 2) identify the signals upstream of SFK phosphorylation of these NMDARs, and 3) investigate the involvement of NMDARs in primary afferents in chronic pain. The approach includes the use of two strains of transgenic mice with selective knockdown of NMDARs in dorsal root ganglia (DRG) to determine whether the NMDARs that induce substance P release (measured as neurokinin 1 receptor internalization) and contribute to neuropathic and visceral pain are located in primary afferents. The SFK that phosphorylates these NMDARs will be identified by selective small interference RNA (siRNA) knockdown in DRG of each of the five neuronal SFKs. Western blots of DRG extracts will serve to measure tyrosine phosphorylation of the NR2B subunit after treatment with BDNF and ephrinB. Patch-clamp recordings in cultured DRG neurons will study changes in NMDARs currents after adding BDNF and ephrinB. Using rodent models, we will test the predictions that neuropathic and visceral pain increase NMDA-induced SP release, and decrease after selective knockdown of NMDARs in primary afferents.